Important Books & Reports

Glyphosate/Roundup, falsely claimed by Monsanto to be safe and harmless, has become the world’s most widely and pervasively used herbicide; it has brought rising tides of birth defects, cancers, fatal kidney disease, sterility, and dozens of other illnesses - more

Ban GMOs Now - Dr. Mae-Wan Ho and Dr. Eva Sirinathsinghji

Health & environmental hazards
especially in the light of the new genetics - more

Living Rainbow H2O - Dr. Mae-Wan Ho

A unique synthesis of the latest findings in the quantum physics and chemistry of water that tells you why water is the “means, medium, and message of life” - more

The Rainbow and the Worm - the Physics of Organisms - Dr. Mae-Wan Ho

“Probably the Most Important Book for the Coming Scientific Revolution” - more

Aptamers for Biosensing, Diagnosis, Drug Delivery and Therapy

A whole new class of molecules make conventional protein antibodies obsolete but are they safe? Dr Mae-Wan Ho

What are aptamers?

Aptamers are making conventional
antibodies obsolete. Aptamers are oligonucleic acids or peptides that bind to
a specific target molecule like conventional protein antibodies but with little
or no immunogenicity and improved stability, and much easier and cheaper to
produce in the test-tube, without the need for cells or animals [1].

Nucleic acid
aptamers, in particular, are finding numerous applications that previously
required the use of antibodies. They are engineered through repeated rounds of in
vitro selection or SELEX (see Box 1) to bind to various targets such as
small molecules, proteins, nucleic acids, even cells, tissues, and organisms.

Box 1

SELEX, an in vitro selection technique for producing
aptamers [2]

SELEX (systematic evolution of ligands by exponential
enrichment) is a method for producing oligonucleotides of single stranded DNA
or RNA that specifically bind to a target ligand. It begins with the synthesis
of a very large oligonucleotide library consisting of randomly generated
sequences of fixed lengths. For length n, the number of possible sequences in
the library is n4, because there are four different bases possible
at each position. The sequences in the library are exposed to the target, which
may be a protein, or a small organic compound. Those that do not bind are
removed. The bound sequences are recovered and amplified by PCR to prepare for
another round of selection in which the stringency of binding conditions is
increased, and so on.

Aptamers were
first produced independently in two laboratories in 1990, that of Larry Gold
[3] at University of Colorado, Boulder, who used the term SELEX (systematic
evolution of ligands by exponential enrichment) for the process of selecting
RNAs binding to T4 DNA polymerase, and Jack Szostak [4] at Massachusetts
General Hospital, Boston, selecting for RNAs binding to various organic dyes,
and coined the terms ‘in vitro selection’ and ‘aptamer’.

The technique
has been used to evolve oligonucleotide aptamers of extremely high binding
affinity to a variety of targets, such as ATP and adenosine, and proteins such
as prion and vascular endothelial growth factor (VEGF). Clinical uses are
suggested for aptamers that bind tumour markers, and a VEGF-binding aptamer
trade-named Macugen has been approved by the FDA for treating macular
degeneration (see below).

Aptamers bind
to ligands based on their three-dimensional structure; hence different
oligonucleotide sequences may recognize the same target molecules.

However, selection for extremely high binding affinities
(at 10-9M or less) does not guarantee absolute specificity.
Off-target binding may have significant, unintended clinical effects [5]. This is particularly important as natural aptamers have been
discovered that play key roles in gene regulation.

Natural aptamers

Natural aptamers were discovered
in 2002 in the nucleic acid genetic regulatory element, the riboswitch
that possesses similar molecular recognition properties to the artificially
made aptamers [6]. A riboswitch is a part of an mRNA molecule – transcribed
from a gene -that directly binds to a small target molecule, usually a
metabolite, to change the gene’s activity [7]. This involves turning off gene
expression via the premature termination of transcription, or inhibition of
translation; or the riboswitch may be an RNA enzyme (ribozyme) that cleaves itself
in the presence of the small metabolite, or it may lead to alternative splicing
of the pre-mRNA. Some riboswitches may even regulate the transcription of
downstream genes; or turn on gene expression as they bind to target
molecules.

Many of the
earliest riboswitches corresponded to conserved sequence motifs in the 5’
untranslated regions of mRNAs. Most known riboswitches occur in viruses and eubacteria,
but they have also been discovered in plants and certain fungi, and predicted
in archaea bacteria. The first human riboswitch was identified in 2009 in human
vascular endothelial growth factor (VEGF) [8]. It is highly likely that
riboswitches may also exist in other animals.

The many uses for aptamers are summarized in Figure 1
[9].

Figure 1 Biological applications of aptamers

Aptamers as antibodies

Oligonucleic
aptamers are replacing conventional protein antibodies because they are much
more stable, easy and cheap to produce, less immunogenic or toxic and can
target a wider range of molecules, including those not giving strong
conventional antibody responses.

Protein antibodies are easily denatured and lose their
structure irreversibly at high temperatures. Oligonucleotide aptamers in
contrast are much more heat- stable, and maintain their structures over
repeated cycles of denaturation/renaturation. Aptamers can therefore be used
under a wider range of assay conditions.

Monoclonal
antibodies are laborious and expensive to produce, requiring large scale mammalian
cell cultures; and different batches have to be tested to ensure that they
maintain the same binding properties. In contrast, aptamers, once selected, can
be synthesized in bulk cheaply with accuracy and reproducibility by chemical
reactions. Aptamers can also be readily modified to increase their stability
and resistance to nuclease breakdown (though this runs the danger that they
will persist in the body and in the environment to do harm), and it is possible
to introduce signalling adducts such as fluorescent molecule and its quencher
for biosensing.

Protein
antibodies are immunogenic, which makes repeated dosing a big problem. Aptamers
are reported to be low in immunogenicity and toxicity, and are readily broken
down in the body, unless modified to prevent breakdown. However, the evidence
for low immunogenicity and toxicity appears to be based solely on the Phase I
and Phase II clinical trials of mucagen [9, 10], which may well not be
generalizable to the entire class of molecules.

Finally, it is often difficult to produce antibodies
against small molecules, ions, and toxins that do not elicit strong immune
response; but aptamers can be generated that bind to those molecules with high
affinity. Aptamers not only substitute for conventional antibodies, but also
have an almost unlimited potential to overcome the limitations of antibodies.

Aptosensors
for food safety, bioimaging and diagnosis

Biosensors
based on aptamers as recognition elements are called aptosensors. Aptosensors
can be made through a variety of techniques; based on changes in
electrochemical potential, fluorescence (see Figure 2), or colour when the aptamer
binds to the target molecule [9]. These aptosensors have found important
applications in food safety, in the rapid detection of toxins and contaminants,
such as antibiotics, pesticides, mycotoxins, heavy metals, bisphenol A, and
adulterants such as illegal food dyes [11].

Aptosensors can also be used in clinical diagnosis in place
of ELISA (enzyme-linked immunoadsorbent assay). In the Aptosensor version
(ALISA), the aptamer is linked to biotin, which, on binding to the target
immobilized on a nitrocellulose filter, reacts with streptavidin and horse
radish peroxidase to generate the colour reaction. This and related techniques
are offering commercially available rapid and simple methods for diagnosis of
infectious diseases such as malaria and influenza [9].

Aptosensor microarrays have been designed that are
particularly good for detecting cell and tissue markers for bio-imaging and
drug delivery. Special light sensitive adduct enable aptamers to covalently
link to bound proteins, making it easy to identify them [12].

Aptamers for specific anti-cancer drug delivery

Aptamers
that bind to internalized cell surface receptors can deliver drugs and other
cargoes into cells. This is very useful in cancer chemotherapy, which aims to
kill cancer cells with cytotoxic drugs without harming noncancerous cells.

The prostate-specific membrane antigen PSMA is an important
marker for prostate cancer. An aptamer combination for PSMA has been used to
deliver doxorubicin, an anticancer drug into prostate
cancer cells [13]. The anticancer drug has also been successfully delivered
into liver- [14] and breast-cancer cells [15] by aptamers that target specific
membrane antigens that are over-expressed in the respective cancer cells. A
novel MUC1 aptamer selectively delivers the cytotoxic agent into both lung and
breast cancer cells [16]; the MUC1 protein is over-expressed in most
adenocarcinomas.

Aptamers are poised to make a big difference to cancer chemotherapy,
which has been notorious for its toxic side effects on account of the
difficulty in selectively targeting cancer cells.

Aptamers
for therapy and new drugs

Macugen,
developed by Pfizer and Eyetech, is already commercially available for treating
age-related macula degeneration, an eye disease that can lead to blindness. The
drug is a polyethylene glycol-aptamer with specificity for VEGF165, which plays
a critical role in angiogenesis (development of blood vessels) and permeability
[17]. Regado Bioscience has developed a new
aptamer drug for anticoagulation currently in Phase II clinical trials. The
drug REG1 consists of two components: RB006 (coagulation factor IXa-specific aptamer) and
RB007 (oligonucleotide antidote of the RB006 aptamer), to control excessive
bleeding due to anticoagulation [18]. In the treatment of acute ischemic stroke
(arising from a decrease in blood supply to the brain), a Factor IXa aptamer was
found to improve neurological function in a mouse model, and treatment with its
specific antidote in cases of intracranial haemorrhage improved survival [19].
These results highlight the need for antidote molecules in a growing number of therapeutic
applications involving aptamers, which may well have off-target effects. Many
are currently undergoing clinical trials, including a nuclein-specific aptamer for
acute myeloid leukemia, a vonWillebrand factor-specific aptamer for carotid artery
disease, and a thrombin-specific aptamer for anticoagulation [20, 21].

Universal
antidote for aptamers

The
potential number of aptamers for therapeutics is unlimited. A number of them target
coagulation, and represent potential anticoagulants. But anticoagulants can
cause complications such as considerable bleeding. Currently only an
anticoagulant and antidote pair, heparin and protamine, is routinely used in
clinics. This is now replaced by an anticoagulant aptamer and its antisense
antidote.

Bruce Sullenger and colleagues at Duke
University, Durham, North Carolina realized that with an ever increasing number
of people taking numerous drugs, the need to safely administer drugs and limit
unintended side effects has never been greater. Between 1998 and 2005, the
number of serious adverse drugs events reported to the USFDA increased 2.6 fold
and fatal adverse events increased 2.7-fold to 15 107 events in 2005.

Antidote control is the most direct way to
counteract acute side effects of drugs, but it has been difficult and cost
prohibitive to generate antidotes for most therapeutic agents. The team
embarked on the development of a set of antidote molecules that are capable of
counteracting the side effects of an entire class of therapeutics – aptamers –
making them a particular safe class of therapeutics.

Previously, the team used Watson-Crick
base-pairing rules to create a customized antidote oligonucleotide for each
aptamer, which is very costly, and also results in a double-stranded RNA
molecule that may stimulate the immune system. Instead, molecules that can
sequester oligonucleotides in a sequence-independent manner should be able to
function as universal antidotes for extracellular oligonucleotide-based drugs.

They screened a number of nucleic acid binding
polymers for their ability to act as antidotes for a series of anticoagulant
aptamers with very different structures. b-cyclodextrin-containing
polycation (CDP) and polyphosphoramidate polymer (PPA-DPA) were found to act as
sequence-independent antidotes for aptamers both in vitro and in vivo,
in mice, and in pigs for CDP, without toxicity, and at lower level of 2.5mg/kg,
compared with the conventional antidote protamine at 10mg/kg.

To conclude

Nucleic
acid aptamers appear to offer infinite opportunities for diagnosis, biosensing,
bio-imaging, drug delivery, therapy, and much more. But key questions on safety
remain largely unanswered. Although unmodified nucleic acid aptamers seem to be
non-immunogenic and non-toxic, and rapidly degraded; the current trend is to
chemically modify them to resist breakdown, hence increasing their therapeutic
efficacy. But this also means that they are more likely to survive in the
environment with unknown consequences for health and the ecology of our aquatic
systems. This is of particular concern, as natural aptamers have been
identified in numerous species including humans, and are very likely to be
widely distributed in the living world.

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There are 4 comments on this article so far. Add your comment above.

Jerome Ravetz Comment left 18th October 2012 09:09:48"they are more likely to survive in the environment with unknown consequences for health and the ecology of our aquatic systems."
This comes in as an afterthought. So even for i-sis our ignorance is still less important than our knowledge.

Mae-Wan Ho Comment left 18th October 2012 11:11:49Hi Jerry,
Sorry it seems like an afterthought to you. But definitely not! I see an obligation to inform on what is known as far as possible and then point out the areas of our ignorance. You would not want me to speak from a position of ignorance do you?
Regards, maewan

Linus Hollis, ScD Comment left 20th October 2012 09:09:55Prion treatment! Obvious after saying it, true, but reversing the RNA cascade with aptamers should be studied asap! RE environmental contamination: if proven, treatment centers with biowaste controls will add costs, but not terribly high, while the life-saving possibilities are devoloped. Bravo!

Jerry Ravetz Comment left 19th November 2012 16:04:10Sorry - I didn't want to be negative about your work, of course.
It's just that in your article the warning came at the very end. This seemed too similar to what one would expect in a mainstream publication, where 'absence of proof of harm is proof of absence of harm'. All the very best - Jerry